The beneficial effect of Allium Cepa bulb extract on reproduction of rats; A two-generation study on fecundity and sex hormones

Allium Cepa Linn. (Onions) has extensively been used in traditional medicine, is one of the important Allium species regularly used in our daily diet, and has been the source of robust phenolic compounds. The current study is intended to evaluate the fecundity-enhancing effect of A. Cepa on the reproductive performance of two successive generations of rats; F0 and F1. A. Cepa extract was initially tested for in vitro antioxidant assay via DPPH and ROS, followed by in vivo toxicity testing. In the fecundity assessment, eighteen pairs of male and female rats (n = 36, 1:1, F0 generation) were divided into three groups and dosed with 75mg/kg and 150 mg/kg daily of A. Cepa extract and saline respectively, up to pre-cohabitation, cohabitation, gestation and lactation period. The reproductive performance, including body weight, live birth index, fertility index, and litter size, was assessed. Various parameters like Hematological, Hormonal (FSH, LH, Testosterone, estradiol), antioxidant markers (SOD, Glutathione peroxidase) and lipid profile of F0 and F1 generations were assessed with evaluation of histopathology of male and female organs. Ethanolic extract of A. Cepa showed the greatest antioxidant potential in DPPH and ROS methods. The continued exposure of the F0 and F1 generations to A. Cepa extract did not affect body weight, fertility index, litter size, and survival index. However, semen pH, sperm motility, sperm count, sperm viability, and semen volume were significantly improved in both generations. We have found pronounced fecundity outcomes in both genders of F0 and F1 generations with A. Cepa 150mg/kg/day extract as compared to control. Results showed that A. Cepa significantly increased (P < 0.05) hemoglobin, follicular stimulating hormone (FSH), luteinizing hormone (LH), plasma testosterone and glutathione peroxidase activities, while total lipid, LDL, and cholesterol were significantly decreased (P < 0.05) in both generations. Histology of both generations of animals reveals enhanced spermatogenesis and enhanced folliculogenesis with improved architecture. Altogether, the present results suggest that A. Cepa extract improved fecundity in both male and female rats by improving hormonal activities and oxidative stress.


Introduction
Infertility can be defined as the failure to accomplish a pregnancy clinically after 12 or more months of regular unsafe sexual intercourse [1].It has been estimated that around 15% of couples are infertile globally [2].Within all infertility issues, human sterility attributed to male fertility issues reports for 45-50% [3], and female accounts for 46.6% of cases with polycystic ovarian syndrome (PCO S ) constituting the principal cause [4].To date, factors underlying the dysfunction of the reproductive system are incompletely understood.The use of recent drugs to improve fertility dysfunction is insignificant in achieving her pregnancy outcomes [5].So, the future focus will be on formulating more viable treatment choices to prevent or treat reproductive system dysfunctions to improve fecundity.
For several years, locally grown plants in place of supplements have been used globally to vitalize, energize, and enhance male sexual functions.Several plants extract have now been identified for improving fecundity and other sexual functions, including Parkia Biglobosa [6], Eugenia Uniflora [7], Montanoa Tomentosa [8], Bryonia Laciniosa [9], Terminalia Catappa [10], Tribulus Terrestris [11] and Lepidium Meyenii [12].These plants' active constituents, including terpenoids, phenols, alkaloids, and saponins, may have a role in restoring fertility [13].Polyphenols, especially flavonoids, are recognized to have estrogenic [14] or androgenic capabilities [15] that have a crucial role in maintaining fecundity.Such findings pointed out that flavonoids can be essential in recovering spermatozoa and spermatogenesis [5].Sperm infertility may be caused by various factors among which ROS over production constitutes a major risk factor.Enhanced ROS production oxidatively spoil almost all biological molecules in cell involving DNA leading to enhanced sperm membrane damage, declined motility and disability of sperm to fertilize the oocyte [16].
Allium Cepa Linn.(Onion) is the well-known edible bulb of the Alliaceae family and is considered among the oldest cultivated species in the world [17,18].It contains various constituents [19] studied because of their different pharmacological properties [20].A. Cepa has been a valuable medicinal plant for decades due to its robust free radical scavenging potential [21].It contains antioxidants such as vitamins, selenium, glutathione, and flavonoids like isorhamnetin and quercetin [22,23].It has been documented that incorporating antioxidants in the diet protects sperm DNA against oxidative damage and improves sperm health [24].Moreover, Ghasemzadeh et al., [25] reported that A. Cepa ethanolic extract administration significantly compensated the blood antioxidant level in the experimental polycystic ovary (PCO) model of Wistar female rats.Recent researchers claim that the presence of these anti-oxidants in A. Cepa may be beneficial and protective in reproductive functions [26][27][28].
Adeleye et al. [29] reported that A. Cepa has increased the serum levels of some hormones, luteinizing hormone (LH), and follicle-stimulating hormone (FSH); it has also improved sperm motility in birds.Another study elucidated the protective effect of A. Cepa juice against dexamethasone-induced oxidative stress during lactation in rats [30].The cadmium-induced sperm toxicity and testicular oxidative stress were successfully attenuated after the administration of A. Cepa aqueous extract to rats [31].The evidence shows that A. Cepa has a protective potential against testicular oxidative damage and reproductive dysfunction by stimulating the free radical scavenging system and decrementing lipid peroxidation.
A. Cepa extract presented its exclusive actions by augmenting fecundity power and diminishing fecundity-related problems.Assessment of Body weight and blood parameters provide information regarding the health of the rodents and reproductive effects of A. Cepa extract [32].However, no research has been executed to explore the beneficial role of A. Cepa on reproduction in two generations.Therefore, a two-generational study was established to explore the beneficial and protective use of A. Cepa extract on fecundity and reproductive hormones in experimental animals.

Extraction of plant material
A. Cepa was purchased from a local market in Karachi, Pakistan, and was distinguished at Herbarium and Botanic Garden, University of Karachi and has got an identification number; G.H. No. 956008 (Allium Cepa L. of Alliaceae family).
The A. Cepa bulbs were washed with distilled water.The outer layer of the bulbs was peeled off manually and rinsed with sterile distilled water, then air dried properly, cut into small pieces, and chopped.The pieces were soaked in 96% ethanol for five days (100 grams of the sample soaked in 200 ml ethanol) at room temperature (25˚C to 30˚C) with intermittent shaking.The ethanol extract was filtered and later evaporated to dryness at 20˚C using a rotary evaporator (Buchi Rotavapor, Germany) and stored at 2˚C-8˚C in a refrigerator [33].

Total phenolic content analysis
The method used for total phenolic content described previously by Lee et al. [37] utilizes Gallic acid as a standard to obtain a curve by plotting.With the aid of serial dilutions utilizing deionized water, different concentrations of dilutions; 50.0, 25.0, 12.5, 6.2, and 3.1 mg/ml, were formulated.Constituent volumes were added to a single vessel: reagent 100 μL: Folin-Ciocalteu; 20 μL: sample-blank (DI water)-standard gallic acid; 80 μL: Na 2 CO 3 (30 g/L).The microplate was allowed to incubate for 15 minutes at room temperature, followed by taking an absorbance reading at 540 nm utilizing a UV-VIS spectrometer (Genesys™ 10S UV-Vis, Thermo Scientific, USA).Dilutions were executed on the extract as considered necessary for the values of absorbance to fit among the standard curve.The results were declared as mg/g of gallic acid equivalent.

Antioxidant assays
Antioxidant assay of A. Cepa by DPPH.An upgraded DPPH assay (1,1-diphenyl-2-picrylhydrazyl) was selected to evaluate the tested compound.The DPPH radicals diminish at 517 nm of absorbance in the presence of antioxidant potential.The test compound A. Cepa at different concentrations (10,20, 100, 300 μg/ml) was added to 0.1mM DPPH (3ml) and allowed to react.The mixture was swirled and incubated for 30 min at room temperature.A microplate reader (SkanIt Software 5.0 of RE, ver.5.0.0.42) was utilized to measure the absorbance at 517 nm.The test compound reducing power was calculated with the absorbance of ascorbic acid, which served as control [38].
Radical Scavenging Activity was equated in percentage via equation.
Antioxidant assay of A. Cepa by ROS.Primary cortical cells were used for the determination of Reactive Oxygen Species (ROS) generation.The method [39] was adopted with some modifications.The untreated cells were used as a control.Cortical cells were harvested from the brain of two-day-old rat pups.Cells were grown in DMEM affixed with 10% FBS and then 1% penicillin-streptomycin (100 units/mL-100 μg/mL) respectively.Adhesive cell monolayers were cultured into T25 tissue culture flasks to be used for further experiments.The cortical cells were incubated at 37˚C in a humidified atmosphere having 5% carbon dioxide (CO 2 ).ROS Intracellular levels of living cells were assessed quantitatively by employing a fluorescent probe, 2 0 ,7 0 dichlorodihydrofluorescein diacetate (DCF-DA).Primary cortical cells were subjected to various concentrations of extract for 12 h.Untreated or treated cultures were allowed to incubate in the dark with 10 μM DCF-DA at 37˚C for 45 minutes, and later on, washed with PBS.Relevant ROS level changes intracellularly were observed by fluorometric detection of DCF by using a fluorescent Microplate Readers RE, ver.5.0.0.42 at emission and excitation wavelengths of 530 nm and 485 nm, respectively.The fluorescence intensity level of DCF is proportional to the ROS level generated intracellularly.Percentage Cell Viability can be calculated by formula [40].
Experimental animals.In the experimental fecundity study, we used 36 healthy animals comprising 18 males and 18 females Wistar rats.The animals were bought from the animal house, H.E.J, University of Karachi, and were kept in plastic cages having proper ventilation under standard temperature conditions (25˚C to 30˚C) with 12 h light and dark cycle.The animals were given pelleted rodent food.The weight of animals ranged between 170g to 190g.The animals were placed in an animal house at the Department of Pharmacology, University of Karachi.
The experimental protocol fulfills the guidelines regarding the appropriate use and care of laboratory animals [41], and the experimental protocol and design were permitted by the Board of Advanced Studies and Research (BASR), Resolution No. 10(P)07, ASRB/No./05071/Pharm., University of Karachi.
Animal ethical approval.The study was performed under the approval of the Board for Advanced Studies and Research dated 28-03-2019 (reference No 05071/pharm), University of Karachi and Pharmacology Departmental Research Committee, for the use of animals according to the National Institute of Health guidelines (NIH guidelines Islamabad, Pakistan) [42].
Brine shrimp toxicity testing.Toxicity of A. Cepa was evaluated to rule out % Mortality with the help of the Brine shrimp method (Artemia Saline).Etoposide served as a positive control [43].
Acute oral toxicity.The study on acute oral toxicity was executed according to the OECD-404 guidelines [44].Both genders' rats were used and supplied with distilled water and fed with adlibitum.Twenty rats were randomly divided into four groups (n = 5).A single oral dose of ethanolic extract of Allium Cepa, 300 mg/kg, 600 mg/kg, and 1200 mg/kg were administered as per body weight respectively, to each group in comparison to the control.The rats were examined for toxicity signs like behavioral changes and mortality for 48 hours [45].
Fecundity assessment.The study was conducted for a total duration of seven months.A modified method of Vohra et al. [46] was used.Eighteen pairs of rats (n = 36, F 0 generation) were placed in separate cages.
In the fecundity study, healthy and sexually mature male rats aged 12 weeks (155.42 ± 1.89 g) and female rats aged 14 weeks (169.1 ± 2.16 g) were used.Rats were isolated and evaluated for any gross symptoms of injury or disease.After one week of acclimatization, six experimental groups were formed.Three groups were of male rats, each comprising of six rats (n = 6), while the rest of the three groups comprises of female rats only (n = 6).
In male rat groups, Group, I served as control and administered distilled water daily; Group II animals were given low dose A. Cepa extract of 75 mg/Kg/day while Group III animals were given A. Cepa extract at the high dose of 150 mg/Kg/day [47,48].A similar protocol was followed for the female group as well.All rats were dosed for 30 days before mating (Pre-Cohabitation) by gastric gavage between 14:00 and 16:00 h.Female rats were subjected to the soiled bedding of an adult male rat to align their estrus cycle (female rats were fertile and receptive during this period) [49].During mating each male rats were individually paired with female rats of each group in separate cages for seven days (1:1).Inseminated females were separated from males and placed in cages after conception.The doses of A. Cepa were given continuously to female rats during cohabitation (21 days), gestation (21 days), delivery and lactation till postnatal day of 22.When pups were delivered, all F 0 animals (male and female) were deeply anesthetized with intraperitoneal Ketamine (10 mg/Kg) and Xylazine (5 mg/Kg) [50], to alleviate the suffering of rats.Blood samples were taken from the aorta and the animals were sent to necropsy.Subsequently, reproductive organs were taken for histopathological assessment.
Fertility indices (fertility index, the number of pups, survival index) were estimated.Offspring were counted and observed for any abnormality on postnatal days 0, 4, 7 14, and 21.Body weight was recorded weekly before, during mating, and in gestational periods.All F 0 male/female parental animals were monitored twice daily for toxicity signs.
For male rats' fecundity assessment, the procedure described by Quadri & Yakubu [51] was selected to prepare the semen from the epididymis.The Epididymis was carefully detached and lacerated with scissors from the testis.The semen was aspirated 10 μl from the caudal epididymis of every rat by using a capillary tube and transferred into Petri dishes already having 1 ml, 0.1 M phosphate buffer having 7.4 pH.The homogeneity was maintained by slightly swirling each dish.The sperm cells were permitted to separate in the solution at 37˚C for 10 min.Afterward, the semen volume, sperm count, pH, motility, and viability were estimated using standard methods.
For F 1 generation study, pups were grown up to 4 weeks then the same doses of the A. Cepa extract were given to 4 weeks old pups (according to their bodyweight) during their growth, adulthood, and up to breeding.After one month, 8 weeks old rats were permitted mating by making pairs on a 1:1 basis within same treatment group, preventing sibling-mating, for 7 consecutive days.The vaginal plug was noticed to confirm the pregnancy of each female rat.
Pregnant female rats of the F 1 generation were separated from male rats and placed individually.A. Cepa extract continuously given during their pre-cohabitation, cohabitation period, gestation, and lactation until the F 2 generation pups were weaned.Gestated female rats were permitted to naturally deliver pups (F 2 generation).
Afterward, all F 1 generation animals were anesthetized and examined as described previously.The same protocol of the F 0 generation was followed for both F 1 and F 2 generation till the birth of F 2 pups.For all F 1 generation female rats' fecundity assessment, the above procedure for determining fertility indices in F 0 generation animals were used as described by Vohra et al. [46].Similarly, for all F 1 generation male rats' fecundity assessment, the procedure described by Quadri & Yakubu [51] was selected as discussed above in F 0 generation animals.
Reproductive performance of F 0 and F 1 rats.The reproductive parameters studied were mentioned in the form of ratios, weights, and indices that analyze all stages starting from conception till weaning [46].These fertility parameters were computed as follows: Fertility index (%) = (Females count giving birth/females count lived together) × 100 Litter size = count of pups/count of gestated females Live birth index (%) = (count of pups alive at day 0/count of pups born) × 100 Survival index-4 days (%) = (count of pups alive on day 4/ day 0 alive pups count) × 100 Survival index-21 days (%) = (count of alive pups on day 21/ day 4 alive pups count) × 100 Blood parameter assessment.When pups were delivered, all F 0 and F 1 generation male and female animals were anesthetized with Ketamine and Xylazine.Blood samples were taken from the aorta for biochemical assessment and kept in B-Ject gel clot activator vacuum tubes under aseptic conditions.Samples were centrifuged in an Eppendorf machine at 4000 rpm for 10 min.The separated serum was kept at -80˚C for hormonal and biochemical testing.All samples were analyzed for hematological (Sysmex KX-21 hematological analyzer) [52], Total lipid profile (LDL, VLDL, HDL, Triglycerides, Cholesterol), hormonal estimation (FSH, LH, testosterone and estradiol [53,54] were assayed via ELISA Roche Diagnostics, Basil).SOD and Glutathione (GPx) were estimated by the kit acquired from Glory Science Co., Ltd.(Glory Science Co.2022#45).
Histopathological evaluation.For histopathological evaluation, the ovaries and testis of the selected rats were removed and cleaned from adherent connective tissues, then fixed in 10% formalin.Fresh tissue samples were dehydrated by exposing ethanol and dimethyl benzene of different concentrations (twice) at room temperature.Then, sex organs were embedded with paraffin at 60˚C and cut into sections.Blocks of paraffin were cut serially and longitudinally at 4 μm thickness, followed by Hematoxylin-Eosin staining.Lastly, the stained sections were dehydrated and sealed with coverslips and imaged under microscope (Bio Base XS-208, China).Stained slides were analyzed and compared in groups [55].

Statistical analysis
Statistical analysis was implemented using the SPSS software (version 20.0).Multiple groups were compared by one-way analysis of variance applying the Bonferroni post-hoc test.The results were considered statistically significant if the value lies between P < 0.05 and highly significant when P < 0.01.

% Yield and total phenolic content of A. Cepa bulb
A. Cepa bulb (12 Kg) was extracted with 96% ethanol and the total yield percentage of A. Cepa extract was found to be 10%.
Total Phenolic Content was determined in the extract of A. Cepa bulb by the method of Folin-Ciocalteu (F-C) using gallic acid as the standard.Absorbance values were determined at various concentrations of gallic acid to construct the calibration curve.The total phenolic content of the extract was estimated from the regression equation of the calibration curve (Y = 1.012x;R 2 = 0.996) designated as gallic acid equivalents (GAE) (63 mg/g GAE) (Table 1).
Acute oral toxicity.Regarding the safety of A. Cepa L. plant, acute toxicity study disclosed no signs of toxicity with no mortality in rats.However, some dose-dependent behavioral changes were observed in high-dose (1200 mg/kg) treated groups, including depression, clustering, folding and sleeping, unsteady gait, and loss of appetite.The results regarding Acute toxicity (LD 50 ) of A. Cepa, by using Karber Method, is mentioned below (Table 2).The arithmetic method of Karber was utilized for calculation.

Preliminary Phytochemical Screening
The presence of A. Cepa constituents was mentioned in Table 3.The qualitative phytochemical analysis reveals that Allium Cepa possesses tannins, flavonoids, terpenoids, glycosides, saponins, phenols, and phlobatannins.

Antioxidant assays
Antioxidant Assay of A. Cepa by DPPH.The % inhibition of A. Cepa extract at different concentrations is shown in Table 4.The extract was observed with radical scavenging potential by DPPH utilizing Ascorbic Acid as a reference standard.Among various concentrations, A. Cepa exhibited the highest activity at 100 μg/ml and 300 μg/ml, i.e., 84.92 ± 0.11% and 80.27 ± 1.20%, respectively, while Ascorbic acid exhibited the strongest scavenging activity of 88.14 ± 1.28% at 300 μg/ml.
Antioxidant Assay of A. Cepa by ROS.The DCF-DA method was employed to determine the ROS production and % Viability of A. Cepa extract on primary cortical neuronal cells was found nontoxic and significantly prevented H 2 O 2 -induced oxidative damage.% Cell viability in neuronal cells after incubation using indicated concentrations of A. Cepa is shown in Table 5, Fig 1, which shows no cytotoxic effect in neuronal cells at concentrations up to 50  (μg/ml).However, at the dose of 100 (μg/ml), A. Cepa extract shows protective efficacy against H 2 O 2 -induced toxicity on neuronal cells.
Brine shrimp lethality testing.The A. Cepa extract was assessed for brine shrimp lethality studies and the results were mentioned in Table 6.No significant toxicity to brine shrimp Artemia salina was found with A. Cepa extract at different concentrations.The A. Cepa extract shows significant results, which represent that the extract is biologically safe.The % mortality of A. Cepa at 1000 μg/ml concentration is 16.66% compared to standard Etoposide, which shows 100% mortality at 1000 μg/ml, indicating the extract is significant and safe for use.
Fecundity assessment.The two-generation fecundity study was conducted for seven months that showed no congenital abnormalities in any of the pups of all the groups of F 0 and F 1 generations.The body weights of F 0 and F 1 animals are mentioned in Table 7, representing insignificant differences among generations and control.The number of pups was counted in each generation and noticed for congenital abnormalities.In all groups, no congenital abnormality was observed.The number of pups increased significantly in the first and second generations compared to the control group (df 5, 30; F 5.51; P < 0.05).
Post Hoc analysis through the Bonferroni test revealed that high-dose administration of Allium Cepa produced statistically significant differences in the number of pups in both generations compared to low dose group and control group rats, as shown in Table 8.
Reproductive performance.The reproductive performance was assessed by observing several parameters, including live birth index, fertility index, and litter size.Insignificant changes were found in all treated groups' live birth, fertility, and survival indexes (P > 0.05).All these parameters were improved in all treated groups, just like the control group; however,  a significant increase in litter size was noticed in the treated group in both generations (P < 0.05) when compared to the control group, as shown in Table 9.
Additionally, in male rats, parameters like semen pH, sperm motility, sperm count, sperm viability, and semen volume are crucial indices of male fecundity as they are key markers in epididymal maturation and testicular spermatogenesis.All these parameters were significantly improved (P < 0.05) in all treated groups when compared to the control group, as shown in Table 10.
Histopathological evaluation of ovaries.Control ovaries of F 0 and F 1 generation rats showed the external cortex surrounded with intact capsule having profuse stroma.A lot of blood vessels parted by unattached connective tissues in the medulla were also observed.The histological ovarian architecture of F 0 and F 1 generation treated with low-dose A. Cepa, animals was similar to controls.However, some primary follicles surrounded beneath the tunica F 0 presents Parent Generation, while F 1 presents 1 st Generation, F 2 presents 2 nd Generation, T 1 shows low dose group while T 2 shows high dose group.n = 6.
Mean ± SEM *P < 0.05 significant ** P < 0.01 highly significant as compared to control. https://doi.org/10.1371/journal.pone.0294999.t010 albuginea of an intact primary oocyte can be seen in low-dose treated ovaries, and degenerative changes were also detected in follicles.F 0 and F 1 generation's animals of high-dose onion treatment showed Graafian follicle and zona pellucida along with the Nucleus of oocytes.Preantral, small antral, and large antral follicles and corpus luteum of different stages were observed.The number of follicle counts was significantly raised in the F 1 high-dose treated animals as compared to other groups.Folliculogenesis was seen in all samples as shown in Fig 6.Histopathological evaluation of testes.Microscopic examination of control group testes of F 0 and F 1 both generation figures showed an architecture of tubular seminiferous epithelia present normally with occasional Sertoli cells and spermatogenic cells at the interstitial area and base of the tubule having negligible connective tissues at the tubular surroundings.
Microscopic examination of low-dose treated testes of F 0 and F 1 generation showed the normal seminiferous tubules having the normal arrangement of Sertoli and spermatogonial cells resting over intact basement membrane.Similarly, in F 0 and F 1 generation's testes, high dose A. Cepa extract showed normal architecture of seminiferous epithelia of tubules along with Sertoli cells and spermatogenic cells having minimal connective tissues at the tubular surrounding.However, nearer to the tubule's central part, primary and secondary spermatids at the center of the seminiferous tubule lumen were observed.Increased production of spermatids and spermatozoa was found as compared to the control and low-dose A. Cepa group as shown in Fig 7.

Discussion
The therapeutic benefits of A. Cepa have been acknowledged since ancient civilizations.Important benefits under investigation nowadays include its effect on fecundity and reproduction [23].Countless experimental researches were conducted to explore the reproductive potential of different herbal extracts to overcome infertility and related problems [7,51].A. Cepa is one of the herbs that have not been investigated widely for its fertility potential, only a few articles are being reported [26,56].This two-generation study was established to evaluate the role of A. Cepa extract in fecundity and the reproductive system.
In the current study, ethanolic extract of A. Cepa exhibited the presence of different phytochemicals, including tannins, flavonoids, saponins, terpenoids, and polyphenols as well as quantitative data suggest the presence of 63mg/g GAE total polyphenolic content.These findings are in accordance with Prakash et al., 2007 [57], who reported the presence of a rich amount of polyphenols in A. Cepa extract with promising antioxidant activity.Antioxidant activity of A. Cepa extract is evaluated by DPPH that showed the highest % inhibition at 100 μg/ml (84.92%).Similarly, in ROS method A. Cepa extract showed protective efficacy against H 2 O 2 -induced toxicity on neuronal cells.These results suggest dose-dependent radical scavenging activity against DPPH and superoxide anion radicals, which agrees with an earlier study [58].
In screening natural products for pharmacological activity, evaluating the toxic characteristics of medicinal extracts is usually an initial step.During such evaluations, the therapeutic dose determination and mortality rate is the initial step to be conducted [59].In the Brine shrimp lethality test, the % mortality of A. Cepa was found at 16.66% at a maximum concentration, which shows that it is safe and significant to use.Similarly, the result of the oral acute toxicity test of A. Cepa ethanolic extract in-vivo showed that it has a very high safety range when administered orally, even at a maximum dose of 1200 mg/kg.According to Pitchaiah et al. [60], there were no mortality and gross behavioral changes in mice fed with A. Cepa extract, which suggested its safety in the acute study.
Body weight assessment provides information regarding the health of rodents [32].The observations showed an insignificant decrease in body weights of F 0 and F 1 females following the treatment with lower (T 1 ) and higher (T 2 ) doses of A. Cepa.The reduction in body weight may be because A. Cepa has anti-obesity properties, thus being involved in reducing body weight and food intake in rats [61,62].But after week III, the body weights of females of both generations gradually increased during the gestation period [46].This finding is in accordance with Mirabeau and Samson [63], who reported immune boosting and weight-improving capabilities of Allium Cepa.
Our data demonstrated increased pups in both F 1 and F 2 generation during the treatment with both doses of onion extract.This indicated that A. Cepa presented its beneficial actions by augmenting the fertility power of the female rats [26][27][28].There was an increase in the litter size of treatment groups in both generations when compared to the control group.Khaki et al. [64] also reported increased reproductive performance after administering A. Cepa juice.In F 1 off-springs, A. Cepa treatment caused a slight decrease in the live birth index of the high dose group when compared to control.No effects were observed on mating indices, viability index, and fertility indices on days 4 and 21.However, the live birth index was increased in the F 2 generation.The outcomes of our study are endorsed further by the investigation of Ara et al. [65], who demonstrated that A. Cepa extract administration prevented tartrazine-induced noxious infertility in male and female mice.That could be due to polyphenolic compounds present in A. Cepa, especially quercetin that can repair oxidative damage and hormonal irregularities [66].
Furthermore, in the current study, we have found increased sperm count, volume, viability, and sperm motility by administration of A. Cepa.This improved result could be due to the antioxidant potential of A. Cepa, that is a rich source of endogenous and exogenous antioxidants like isorhamnetin and glutathione.It also holds trace minerals and different vitamins; A, B, and C that can assist in preventing damage by reactive oxygen species and protect the tissue from oxidative damage.All these effects may lead to enhanced fertility in males [65].
In our bi-generational experiment (F 0 , F 1 ), blood parameters were normal besides platelet count, which increased in both treatment groups.Therefore, A. Cepa demonstrated potential benefits in maintaining the animal's good health [67].A study also reported that onion extract has blood lipid levels reducing potential [68].Thus, we can conclude that Allium Cepa can efficiently decrease the health risk of cardiovascular events in both genders as it is rich in fiber and flavonoids [69,70].
Reproductive hormones are essential in the development and regular functions of the reproductive system.FSH and Testosterone are important for fulfilling reproductive abilities in males [71].Studies showed that sex hormones like FSH, LH, and testosterone levels are associated with spermatogenesis [64].LH activates Leydig cells to produce testosterone while FSH encourages Sertoli cells proliferation.Both these hormones affect testosterone levels that is necessary for sustaining the maturing sperm cells.Moreover, FSH and testosterone impacts the release and growth of spermatids.Thus, an appropriate concentration of FSH, LH and Testosterone is desired for spermatogenesis [72].
FSH, LH, testosterone, and Estradiol levels were estimated in both F 0 and F 1 generation animals (male and female rats).In male rats, significantly increased effects were observed by A. Cepa (150 mg/Kg) on FSH, LH, and Testosterone levels in both generations, depicting its potential as a fertility-enhancing agent.This result is in accordance with the study of Banihani, who reported that A. Cepa extract consumption enhances testosterone production in males [73].Testosterone is vital for the maturation and maintenance of the structure and function of the male accessory sex glands.Moreover, insufficiency of this hormone hinders spermatogenic function [74].Further, Banihani et al. suggested the mechanisms by which A. Cepa enhances testosterone production in males are mainly by enhancing the antioxidant defense mechanism (e.g., antioxidant enzymes, glutathione) in the testis, ameliorating insulin resistance and promoting nitric oxide production in Leydig cells [73].
Increased effects were observed on FSH levels in female rats (F 1 generation), while in F 0 generation, FSH levels were insignificant.Additionally, estradiol levels were insignificant and maintained as in the control group.Moreover, LH levels were maintained normal, indicating LH potentiates follicle-stimulating hormone (FSH), stimulates follicle growth, and improves female fertility [75].Since FSH/LH surge is required for ovulation that is triggered by LH releasing hormone, dependent on estradiol levels [76].Our results provide the rationale that A. Cepa in higher doses significantly improved FSH levels by balancing the estradiol and improving fecundity, probably due to the increased pituitary sensitivity to gonadotropinreleasing hormone [77].These findings are in accordance with Mansouri et al., who explained that A. Cepa extract significantly improves sex hormone production and fertility in electromagnetic field-exposed mice [78].The changing patterns of sex hormones in the present study are in line with the findings of earlier studies, as Adelakun et al. [79], reported that natural extracts have flavonoid contents that decrease oxidative stress and have pro-fertility properties, which are beneficial for fertility.
Recent studies have reported the anti-oxidant properties of A. Cepa are due to flavonoid quercetin [80].Quercetin-induced upregulation of glutathione levels may elucidate the antioxidant effects of Allium Cepa [81]. A. Cepa extract increased the level of antioxidants in the body, such as glutathione [82].Our study also confirmed the anti-oxidant potential of A. Cepa extract by increasing the glutathione (GPx) levels of both male and female Wistar rats.Present findings for glutathione (GPx) depicted the highest correlation between antioxidant potential (DPPH, ROS) and total phenolic contents in A. Cepa extract.
In Histopathological examination, female rats exhibited an improved number of follicles count as well as Folliculogenesis in the F 1 high-dose treated animals.In addition, the male testis showed improved architecture of seminiferous tubular epithelia along with Sertoli cells and increased spermatogenic cells.These all above results are in line of agreement with Ara et al. [65], who reported that onion extract significantly ameliorated tartrazine-induced histopathological changes with improved architecture, granulosa cells, and corpus luteum.This could be due to the antioxidant activity of A. Cepa extract against reproductive deteriorations.

Conclusion
In conclusion, this study revealed that administration of A. Cepa ethanolic extract for seven months had beneficial effects on both male and female fecundity processes by enhancing ovulation and spermatogenesis with no toxic effect in both sexes.This could be related to its lipidlowering action as manifested by the modulation of serum total cholesterol as well as its antioxidant and androgenic effects.Therefore, it is suggested that daily consumption of A. Cepa may provide an effective strategy to improve fertility.However, further infertility induced model studies are needed to investigate the effects of its active constituents and pure compounds in clinical trials.

Table 9 . Developmental findings in F 1 and F 2 rat pups.
Parent Generation, while F 1 presents 1 st Generation, F 2 presents 2 nd Generation, T 1 shows low dose group while T 2 shows high dose group.n = 6.